Cell based models are extensively used in drug discovery and biomedical research. Cells are typically grown in vitro using two dimensional culture systems or in three dimensions in, for example, expensively engineered bioreactors, solid gels, or hanging drops. Adherent cells attach to the bottom of the cell culture vessels or dishes and remain attached as they grow. In a suspension culture, on the other hand, the cells do not adhere to a fixed substrate, and grow optimally if they float suspended in cell culture medium. Depending on the cell type, these cells may spontaneously form multi-cellular bodies such as spheroids or they may divide rapidly. The advantage of growing cells in suspension is that the cells may either aggregate to form multi cellular bodies or grow and divide freely as single cells. Cells which are grown in conventional suspension culture media passively drift under gravitational force, over very short periods of time, towards the bottom of the culture vessel in which they are maintained. As a consequence these cells are maintained in close proximity to each other at the base of the culture vessel, where at high density, cells experience reduced oxygen and nutrient availability, and elevated levels of metabolic waste products. Consequently, cells growing in this environment may in many cases experience retarded growth rates that can be even further exacerbated by cell-to-cell contact inhibition. This is particularly problematic where it is necessary to grow cells for applications such as the large-scale production of cellular products, for example proteins or glycoproteins.
The passive sedimentation of cells to the bottom of the culture vessel is a widely acknowledged problem with suspension cultures and is dealt with in a number of ways. In small scale culturing facilities, the cell culture medium is changed often to reduce the negative effects of the accumulation of cells at the base of the culture vessel. In larger facilities, where cells are grown in bioreactors, the cells are continually agitated to prevent them from drifting to the bottom of their container. The first solution can be inefficient, labour intensive, expensive, wasteful, and could potentially produce unwanted side effects while the second is expensive and potentially damaging and destructive to the cells and their growth (due to sustained mechanical perturbation of the continuously agitated cells).
When adequately suspended in 3 dimensions, some cell types tend to aggregate into multi-cellular bodies (commonly referred to as spheroids) which in some assay systems resemble tumours found in vivo. These bodies can form either by self-assembly i.e. where cells actively aggregate, by forced aggregation, by cell division, or a combination of all three. Despite the fact that these multi-cellular bodies will not permit rapid growth of cells, if the size and structure of the multi-cellular bodies can be controlled, they represent an extremely valuable research tool due to their physiological relevance in specific research applications. Similarly, pluripotent stem cells may aggregate into multi-cellular bodies known as “embryoid bodies”. While these multi-cellular bodies may also be regarded as spheroidal in nature, they are distinct from the tumour-resembling spheroids previously discussed because they may have the propensity to grow to larger sizes. Due to the intrinsic value of pluripotent stem cells, it is thus clear that the cultivation of embryoid bodies is also an important research goal. It is therefore apparent that effective 3-dimensional growth of cells to produce robust model systems is desirable.
Currently, extracellular matrix solid gels such as Matrigel™ and soft agarose are used to grow cells in three dimensions. Matrigel is a naturally derived growth substance which forms a scaffold similar to the extra cellular matrix, and has been used in culture medium to study tumour spheroids and in the development of in vitro metastasis models. The drawback with Matrigel is that this gel must be in a solid state to grow cells in 3-dimensions. Cells grown in such a viscous environment are not easily analysed by automated high throughput imaging systems, as they are difficult to treat with analytical agents such as cellular dyes, and more often than not, the cells do not lie in a single focal plane. Additionally, sampling secreted substances is technically challenging, and Matrigel is not compatible with laboratory liquid handing devices. As such, the experimental workflows using these types of materials are difficult to automate and hence not readily suited to high throughput screening applications.
Alternatively, engineered scaffolds, such as micro-scale solid structures may be used as inserts or built in to culture dishes or culture vessels. Such structures provide a 3-D scaffold made from materials such as functionalised polystyrene that provide a growth matrix for cells to grow into and for tissue like structures, while at the same time permitting adequate perfusion of nutrient to the cells. These scaffolds are extremely expensive to produce and are only compatible with certain cell types. The scaffold also makes it difficult to image cells and it is not readily practical to extract cells grown using this system.
Also, micro-patterned surface technologies have been used to encourage aggregation of cells. However, experiments using such plates have been restricted to specialized micro-plates which are often only provided in a limited range of designs, densities and materials. Round bottom plates of this type are difficult to image, and the surface patterning or micro-features can further interfere with imaging systems (a common complaint is that these technologies can throw off auto focus). Furthermore, cells are forced together in aggregates using these technologies (rather than allowing the aggregates to form passively), and this technology has also proved expensive to use.
Hanging drop technologies present a further solution as they permit the formation of spheroidal cellular aggregates by bringing cells together in close proximity in a controlled way. These technologies are technically cumbersome, requiring a skilled operator to use, and the imaging of cellular samples within this technology can be difficult without using additional or specialized plates.
Passive cell sedimentation is also a problem in a number of other areas of cell based research. For example, in flow cytometry, cells that sediment out of solution can result in an irregularly dispersed cell suspension that can impact results. Flow cytometry probes have difficulty picking up sedimented cells, and if large bodies of sedimented cells are picked up, this can clog the fluidics of the machine. This is a particular problem in high-throughput flow cytometry, where multiple samples are provided in multiwell plates, and manually re-suspending each sample is laborious and technically challenging. In acoustic droplet ejection (ADE) systems, cells sediment to the bottom of a sample and out of the region of the sample that is to be ejected (with current technologies this is a region close to the bottom of the fluid meniscus), resulting in low numbers of dispersed cells. Effective cell suspension is also desirable in electroporation, where it is necessary to maintain cells in effective suspension during the electroporation process. It is similarly desirable to maintain cells in effective suspension for the purposes of lipid-mediated transfection, or lipofection.